The invention relates to a time-of-flight (TOF) miniature mass spectrometer (MMS), and more particularly to an automated TOF MMS collection, measurement and analysis system for acquisition of mass spectra.
One of the most powerful laboratory tools for analyzing a broad spectrum of chemical and biological material is the mass spectrometer. Mass spectrometry is a proven technique for analyzing many types of environmental samples. Mass spectrometry is used to determine the masses of molecules formed following their vaporization and ionization. Detailed analysis of the mass distribution of the molecule and its fragments leads to molecular identification. Mass spectrometry is especially suited for aerosol analysis because micrometer-sized heterogeneous particles contain only about 10xe2x88x9212 moles of material and thus requires a sensitive technique such as mass spectrometry for proper analysis. Liquid samples can be introduced into a mass spectrometer by electrospray ionization (1), a process that creates multiple charged ions. However, multiple ions can result in complex spectra and reduced sensitivity.
A preferred technique, matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), has become popular in the analysis of biological polymers for its excellent characteristics, such as ease of sample preparation, predominance of singly charged ions in mass spectra, sensitivity and high speed. Time-of-flight MALDI-TOF-MS is established as a method for mass determination of biopolymers and substances such as peptides, proteins, and DNA fragments. The analytical sensitivity of TOF MS is such that under the right conditions only a few microliters of analyte solution at concentrations down to the attomolor (10xe2x88x9212 moles) range are required to obtain a mass spectrum. The MALDI-MS technique is based on the discovery in the late 1980s that desorption/ionization of large, nonvolatile molecules such as proteins can be effected when a sample of such molecules is irradiated after being co-deposited with a large molar excess of an energy-absorbing xe2x80x9cmatrixxe2x80x9d material, even though the molecule does not strongly absorb at the wavelength of the laser radiation. The abrupt energy absorption initiates a phase change in a microvolume of the absorbing sample from a solid to a gas while also inducing ionization of the sample molecules. Detailed descriptions of the MALDI-TOF-MS technique and its applications may be found in review articles by E. J. Zaluzed et al. (Protein Expression and Purifications, Vol. 6, pp. 109-123 (1995)) and D. J. Harvey (Journal of Chromatography A, Vol. 720, pp. 429-4446 (1996)), each of which is incorporated herein by reference.
In brief the matrix and analyte are mixed to produce a solution with a matrix:analyte molar ratio of approximately 10,000:1. A small volume of this solution, typically 0.5-2. microliters, is applied to a stainless steel probe tip and allowed to dry. During the drying process the matrix codeposits from solution with the analyte. Matrix molecules, which absorb most of the laser energy, transfer that energy to analyte molecules to vaporize and ionize them. Once created, the analyte ions the ions formed at the probe tip are accelerated by the electric field toward a detector through a flight tube, which is a long (on the order of 0.15 to 1 m) electric field-free drift region. Since all ions receive the same amount of energy, the time required for ions to travel the length of the flight tube is dependent on their mass to charge ratio. Thus, low-mass ions have a shorter time of flight (TOF) than heavier ions. All the ions that reach the detector as the result of a single laser pulse produce a transient TOF signal. Typically, ten to several hundred transient TOF mass spectra are averaged to improve ion counting statistics. The mass of an unknown analyte is determined by comparing its experimentally determined TOF to TOF signals obtained with ions of known mass. The MALDI-TOF-MS technique is capable of determining the mass of proteins of between 1 and 40 kDa with a typical accuracy of +xe2x88x920.1%, and a somewhat lower accuracy for proteins of molecular mass above 40 kDa. The ability to generate UV-MALDI mass spectra is critically dependent upon the co-crystallization or very close special proximity of the analyte and a molar excess of the matrix compound. In routine practice, a small volume of matrix solution that delivers a one thousand-fold molar excess of matrix is manually mixed with a small volume of the analyte solution which then dries on a sample stage. A spatially heterogeneous distribution of analyte and matrix typically develops as the droplet dries to form a sample spot. Under laboratory conditions, the incident laser is rastered across the sample to identify so called xe2x80x9csweet spotsxe2x80x9d that preferentially yield for an abundance of analyte ions. Although a motorized x-y stage may be incorporated for automated searching for the spot providing the best spectrum, this procedure can be a time consuming step.
MALDI is typically operated as an offline ionization technique, where the sample, mixed with a suitable matrix, is deposited on the MALDI target to form dry mixed crystals and, subsequently, placed in the source chamber of the mass spectrometer. Although solid samples provide excellent results, the sample preparation and introduction into the vacuum chamber requires a significant amount of time. Even simultaneous introduction of several solid samples into a mass spectrometer or off-line coupling of liquid-phase separation techniques with a mass spectrometer do not use TOF mass spectrometer time efficiently.
To improve on these procedures, microfabricated targets have recently been developed for automated high throughput MALDI analysis. In these designs, pL-nL sample volumes can be deposited into a microfabricated well with dimensions similar to the spot size of the desorbing laser beam about 100 micrometers to 1,000 micrometers diameter). Thus, the whole sample spot can be irradiated and the search for the xe2x80x9csweet spotxe2x80x9d eliminated. Analysis of short oligonucleotides has been demonstrated with about 3.3 s required to obtain a good signal to noise ratio for each sample spot. Although the total analysis time, including the data storage, takes nearly an hour, theoretically all 96 samples could be recorded in about five minutes.
While the miniaturization of the sample target simplifies the static MALDI analysis, on-line coupling would allow continuous analysis of liquid samples including direct sample infusion and the monitoring of chromatographic and electrophoretic separations. Compared to ESI, MALDI provides less complex spectra and, potentially, higher sensitivity. There have been numerous reports in the literature about the MALDI analysis of flowing liquid samples. In one arrangement, the sample components exiting a CE separation capillary were continuously deposited on a membrane presoaked with the matrix and analyzed after drying. In other cases, the liquid samples were analyzed directly inside the mass spectrometer using a variety of matrices and interfaces. MALDI was then performed directly off rapidly dried droplets. In another design, a continuous probe, similar to a fast atom bombardment (FAB) interface, was used for the analysis of a flowing sample stream with liquid matrix. Glycerol was used to prevent freezing of the sample. Other attempts for liquid sample desorption were also made using fine dispersions of graphite particles and liquid matrices instead of a more conventional matrices. More recently, an outlet of the capillary electrophoresis column was placed directly in the vacuum region of the TOF mass spectrometer. The sample ions, eluting in a solution of CuCl.sub.2, were desorbed by a laser irradiating the capillary end. On line spectra of short peptides separated by CE were recorded. Attempts to use ESI to introduce liquid sample directly to the evacuated source of a mass spectrometer have also been reported.
Standard MALDI sample preparation techniques as just discussed are not applicable to a real-time TOF-MS systems, the constraints of which do not permit either the analyte and matrix to be mixed in solution or the laser to be rastered across the sample. An additional major design goal of a real-time system is increased throughput speed by avoiding or minimizing the extent to which samples must be processed prior to acquisition of mass spectra. Since MALDI-MS is being used, ideally it is preferred to intimately mix the concentrated sample with a large molar excess of MALDI matrix to produce a uniform analyte-matrix lattice across the sample spot. An alternate technique of depositing an analyte sample in aerosol form directly on a bare collection substrate, or pre-coated surface with a MALDI matrix might not provide the degree of intimate mixing and co-crystallization of the analyte with the matrix that for generation of high quality UV-MALDI mass spectra. Thus, with this second method, additional post-collection steps, e.g., over-spraying with MALDI matrix, may be required.
Another shortcoming of current TOF MS designs are the long pump-down times associated with the introduction of the samples into the vacuum chamber. In the operation of a conventional mass spectrometer a test sample must be introduced through a valve into a vacuum chamber to a location less than a millimeter from an ion extraction source. The introduction of a sample into the MMS vacuum chamber in a real-time system requires rapid sample exchange while maintaining a high vacuum. Current mass spectrometer models require about 5 minutes to pump-down to high vacuum after the introduction of a new sample. A pump-down time of seconds would better meet the requirements of a real-time device.
Although the above-listed examples show efforts to address various different problems related to sample preparation and extraction for a real-time spectrometer, currently there is no-real time device that would permit continuous on-line processing of multiple samples. A device for continuous introduction of individual samples into a time-of-flight mass spectrometer so that on-line MALDI-MS analysis can be carried out would be highly desirable.
In view of the above described state of the art, the present invention seeks to realize the following objects and advantages.
It is a primary object of the present invention to provide a mass spectroscopic analysis system and method which is fully automated requiring no operator interaction.
It is also an object of the present invention to provide a mass spectroscopic analysis system which is portable and reliable enough to survive transport on a range of vehicles, allows handling by two persons, and operates from a portable power source.
It is also an object of the present invention to provide a mass spectroscopic analysis system and method which can carry out spectrographic analysis results faster than previously possible.
It is also an object of the present invention to provide a mass spectroscopic analysis system and method that is suitable for field applications.
It is another object of the present invention to provide a mass spectroscopic analysis system and method which includes provisions for thoroughly mixing an analyte with a matrix composition, thus facilitating real-time spectral analysis.
It is a further object of the present invention to provide a mass spectroscopic analysis system and method which may use, but does not necessarily, require post-collection fluid matrix processing prior to performing a mass spectral analysis.
It is also a further object of the present invention to provide a mass spectroscopic analysis system and method which reduces contamination of the procedure.
It is also an object of the present invention to provide a mass spectroscopic analysis system and method provides a permanent storage medium that has the ability to record pertinent data associated with the collection and measurement of the sample.
It is also a further object of the present invention to provide a mass spectroscopic analysis system and method which includes an external ionization source and electrostatic lens, thus removing the necessity of inserting the sample into the mass spectrometer""s vacuum chamber, thus keeping vacuum pump-down times to a minimum and allowing real-time spectral analysis.
It is a further object of the present invention to provide a mass spectroscopic analysis system and method which promotes rapid throughput and utility of MALDI-TOF MS.
It is also an object of the present invention to capture infectious and toxic agents on a substrate in small spots that allow maximum coverage by an irradiating laser beam. The beam may cover less than about 0.1 mm diameter to greater than 1.0 mm in diameter.
It is another object of the present invention to provide a mass spectroscopic analysis system and method which provides for a variety of techniques for applying and mixing matrix with analyte, thus facilitating real-time spectral analysis.
These and other objects and advantages of the invention will become more fully apparent from the description and claims which follow, or may be learned by the practice of the invention.
As will be appreciated, the present invention provides an automated mass spectroscopic analysis system that may be characterized as an xe2x80x9cend-to-endxe2x80x9d process of sample collection, preparation, measurement and analysis. The present invention is distinguishable from prior art approaches in that conventional approaches are neither integrated nor automated. That is, in the prior art each process is manually performed under operator control and guidance. In accordance with the present invention, a mass spectroscopic analysis systems is provided which performs the following method steps: (1) collect, concentrate, and separate aerosols from breathable ambient air at concentrations on the order of 15 ACPs per liter of air and of 0.5 to 10.0 um aerodynamic diameter. It should be noted that while concentrations on the order of 15 ACPs per liter and of 0.5 to 10.0 um aerodynamic diameter are described, other particle concentrations and densities are also within the contemplation of the present invention; (2) capture infectious and toxic agents from the collected, concentrated and separated aerosols on a continuous substrate (e.g., flexible tape) in small spots that allow coverage by an irradiating laser beam on the order of 1.0 mm in diameter. It should be noted that using a laser with a spot size greater than or less than 1.0 mm in diameter is also within the contemplation of the present invention; (3) prepare the collected samples for the MALDI process by adding a matrix, (4) introduce the collected samples directly into the analysis system in real-time on the continuous substrate. That is, after collection is completed for each sample, the tape transports the sample into a time-of-flight (TOF) mass spectrometer analyzer. The apparatus of the present invention provides a novel vacuum interface which advantageously reduces the vacuum pump loading by isolating the main vacuum chamber from the sample port around the tape sample when samples are being changed. The vacuum interface is formed in part by utilizing the tape as a temporary boundary to form a vacuum chamber seal at or below micro-Torr pressure levels and (5) once inside the high vacuum chamber, a laser than ionizes the sample, and the resulting mass spectrum is analyzed for specific biomarkers that indicate the presence and identity of a biological agent.
The automated system of the present invention provides a number of advantages over prior art approaches including, a minute volume of fluid required for sample processing, eliminating the need for large storage reservoirs, stationary and level mounting configurations, or large power-hungry heating and cooling systems. Further advantages include the concurrent collection of multiple samples, allowing both the application of different analysis protocols and the archiving of samples for later confirmatory analysis.
In practice of the method of the invention, a sample is placed on a permanent storage medium (e.g., a VCR tape) that limits cross sample contamination and undergoes a variation of a matrix-assisted laser desorption/ionization (MALDI) preparation. Each sample is then advanced on the tape to the mass spectrometer analyzer for acquisition of mass spectra. A movable platen forces the tape against a sealing surface, thus creating a vacuum seal with an external vacuum chamber. A triggered laser and an external electric field ion extraction source provides the necessary ionization to initiate mass spectra analysis using a time-of-flight mass spectrometer. When the analysis is complete, the tape advances and a new sample can be analyzed.
Although the analyzer of the invention is achievable in a number of configurations, an acceptable configuration includes: (1) An aerosol interface including a particle collector/impactor stations for collecting, concentrating, and separating analyte from the sample aerosol. A nebulizer for injecting MALDI matrix particles into a sample aerosol upstream of one or more tape particle collector/impactor stations. Continuous tape substrate to collect, hold, and store the analyte and matrix mixture. The nebulizer is preferably automatically controlled to inject metered amounts of MALDI matrix aerosol from the one or more MALDI dispensers into an incoming air stream bearing the analyte to provide thorough mixing prior to collection on a VCR tape. Typically, the aerosol of interest have concentrations of 15 agent containing particles (ACPs) per liter of air and an aerodynamic diameter 0.5 to 10.0 um, (2) a tape transport system for advancing the concentrated samples into a mass spectrum analyzer instrument one at a time for acquisition of mass spectra while continuously and simultaneously collecting new aerosols (samples). The tape transport system includes one or more closed-loop control motors to independently position the tape both inline with the one or more aerosol collectors and with the inlet to the mass spectrometer, (3) a micro applicator may optionally be included to apply MALDI matrix to the samples after collection or to supplement co-deposited matrix to increase sensitivity; (4) a time-of-flight mass spectrometer including an ionization/desorption cell located outside the walls of the vacuum chamber, and (5) a data acquisition system for collecting data, preferably digitized, to be stored in a computing device.
It is noted that it is within the contemplation of the present invention to perform sample preparation by means other than co-deposition, such as, for example, interspersed collection deposition and a post-collection deposition. Other means not explicitly recited herein are also within the scope of the present invention.
Advantages of the apparatus of the present invention include short analysis times (e.g., less than 5 minutes), high sensitivity, wide agent bandwidth, portability, low power consumption, minimal use of fluids required for sample processing thereby eliminating the need for large storage reservoirs, stationary and level mounting configurations, or large power-hungry heating and cooling systems, extending unattended operation, automated detection and classification, and the concurrent collection of that multiple samples allowing both the application or different analysis protocols and the archiving of samples for later confirmatory analysis.